Process for reducing mineralization of tissue used in transplantation

ABSTRACT

A method of making a tissue-derived implantable medical device that includes contacting the tissue with a composition comprising at least one oxidizing agent prior to implantation of the medical device.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to ProvisionalApplication Nos. 60/102,514, filed Sep. 30, 1998, 60/103,697, filed Oct.9, 1998, and 60/105,949, filed Oct. 28, 1998, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The surgical implantation of prosthetic devices (prostheses) intohumans and other mammals has been carried out with increasing frequency.Such prostheses include, by way of illustration, heart valves, vasculargrafts, urinary bladders, heart bladders, left ventricular-assistdevices, and the like. The prostheses may be constructed from naturaltissues, inorganic materials, synthetic polymers, or combinationsthereof. By way of illustration, mechanical heart valve prosthesestypically are composed of rigid materials, such as polymers,carbon-based materials, and metals. Valvular bioprostheses, on the otherhand, typically are fabricated from either porcine aortic valves orbovine pericardium.

[0003] Prostheses derived from natural tissues are preferred overmechanical devices because of certain clinical advantages. For example,tissue-derived prostheses generally do not require routineanticoagulation. Moreover, when tissue-derived prostheses fail, theyusually exhibit a gradual deterioration which can extend over a periodof months or even years. Mechanical devices, on the other hand,typically undergo catastrophic failure.

[0004] Although any prosthetic device can fail because ofmineralization, such as calcification, this cause of prosthesisdegeneration is especially significant in tissue-derived prostheses.Indeed, calcification has been stated to account for 50 percent offailures of cardiac bioprosthetic valve implants in children within 4years of implantation. In adults, this phenomenon occurs inapproximately 20 percent of failures within 10 years of implantation.See, for example, Schoen et al., J. Lab. Invest., 52, 523-532 (1985).Despite the clinical importance of the problem, the pathogenesis ofcalcification is not completely understood. Moreover, there apparentlyis no effective therapy known at the present time.

[0005] The origin of mineralization, and calcification in particular,has, for example, been shown to begin primarily with cell debris presentin the tissue matrices of bioprosthetic heart valves, both ofpericardial and aortic root origin. Bioprosthetic cross-linked tissuecalcification has also been linked to the presence of alkalinephosphatase that is associated with cell debris and its possibleaccumulation within implanted tissue from the blood. Still others havesuggested that mineralization is a result of phospholipids in the celldebris that sequester calcium and form the nucleation site of apatite(calcium phosphate).

[0006] Regardless of the mechanism by which mineralization inbioprostheses occurs, mineralization, and especially calcification, isthe most frequent cause of the clinical failure of bioprosthetic heartvalves fabricated from porcine aortic valves or bovine pericardium.Human aortic homograft implants have also been observed to undergopathologic calcification involving both the valvular tissue as well asthe adjacent aortic wall albeit at a slower rate than the bioprostheticheart valves. Pathologic calcification leading to valvular failure, insuch forms as stenosis and/or regeneration, necessitatesre-implantation. Therefore, the use of bioprosthetic heart valves andhomografts have been limited because such tissue is subject tocalcification. In fact, pediatric patients have been found to have anaccelerated rate of calcification so that the use of bioprosthetic heartvalves is contraindicated for this group.

[0007] Several possible methods to decrease or prevent bioprostheticheart valve mineralization have been described in the literaturesince-the problem was first identified. Generally, these methods involvetreating the bioprosthetic valve with various substances prior toimplantation. Among the substances reported to work are sulfatedaliphatic alcohols, phosphate esters, amino diphosphonates, derivativesof carboxylic acid, and various surfactants. Nevertheless, none of thesemethods have proven completely successful in solving the problem ofpost-implantation mineralization.

[0008] Currently there are no bioprosthetic heart valves that are freefrom the potential to mineralize in vivo. Although there is a processemploying amino oleic acid (AOA) as an agent to prevent calcification inthe leaflets of porcine aortic root tissue used as a bioprosthetic heartvalve, AOA has not been shown to be effective in preventing themineralization of the aortic wall of such devices. As a result, suchdevices may have to be removed.

[0009] Accordingly, there is a need for providing long-termcalcification resistance for bioprosthetic heart valves and othertissue-derived implantable medical devices which are subject to in vivopathologic calcification.

SUMMARY OF THE INVENTION

[0010] The present invention provides a method for reducing the level ofmineralization of tissue in a tissue-derived implantable medical device.Preferably, the method reduces the level of bioprosthetic valvularmineralization, and in particular bioprosthetic vaivular pathologiccalcification.

[0011] In a preferred embodiment of the invention, the tissue-derivedimplantable medical device treated by a method of the invention exhibitsimproved anti-mineralization properties, and/or longer term resistanceto in vivo pathologic calcification than provided by other methods ofreducing and/or preventing mineralization. Although not wishing to bebound by theory, the method of the invention may inhibit enzymes andother proteins (e.g., calcium binding proteins) that are present withinthe tissue from performing their specific functions. These proteins areprincipally involved in phosphate and calcium metabolism and may beimportant in the formation of calcium phosphate, the major component ofmineralized tissue. Treatment by the method of the invention mayeffectively inactivate such protein activity and reduce the accumulationof phosphates and/or calcium in the tissue after implantation, thusreducing the initiation of the mineralization process.

[0012] In another preferred embodiment, the method provides treatmentsperformed on tissue during a method of making a tissue-derivedimplantable medical device. These treatment steps can be performedimmediately upon excision of tissue from an animal, for example, orsubsequent to incorporating the tissue into the device. A preferreddevice is a bioprosthetic heart valve. The method reduces mineralizationon valvular leaflets and supporting structures, such as the aorticwalls, after the device is implanted into patients. Reduction ofmineralization of both valvular leaflets and aortic walls may allow forimproved performance of the device over the duration of the implant.

[0013] In one embodiment, the present invention provides a method ofmaking a tissue-derived implantable medical device. The method involvescontacting the tissue with a composition that includes at least oneoxidizing agent prior to implantation of the medical device. Preferably,the method further includes rinsing the tissue to remove at least aportion of, and preferably substantially all of, the oxidizing agent.Preferably, the tissue-derived implantable medical device is a heartvalve, which can be derived from porcine aortic root tissue, bovineaortic root tissue, porcine pericardium, bovine pericardium, bovineveins, porcine veins, bovine arteries, or porcine arteries.

[0014] Preferably, the oxidizing agent is selected from the group ofsodium hypochlorite, sodium bromate, sodium hydroxide, sodium iodate,sodium periodate, performic acid, periodic acid, potassium dichromate,potassium permanganate, chloramine T, peracetic acid, and combinationsthereof. More preferably, the oxidizing agent is selected from the groupof sodium hypochlorite, performic acid, periodic acid, peracetic acid,and combinations thereof.

[0015] The composition that includes an oxidizing agent preferablyfurther includes at least one chelating agent. Examples of suitablechelating agents include ethylenediaminetetraacetic acid (EDTA),ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), citric acid,salts thereof, and combinations thereof.

[0016] The composition that includes an oxidizing agent also preferablyfurther includes a buffer (i.e., buffering agent). Preferably, thebuffer has a pKa of about 7.0 to about 7.5. More preferably, the bufferis an organic buffer, such as HEPES, TES, BES, MES, MOPS, or PIPES.Various combinations of oxidizing agents, buffering agents, andchelating agents can be used in such compositions.

[0017] In a particularly preferred embodiment, tissue such as porcineaortic root tissue is dissected from the hearts of animals and thenshipped on ice in order to reduce autolytic damage to the tissue and tominimize bacterial growth during shipment. Preferably, either at thesite of slaughter or shortly thereafter prior to significant tissuedamage and/or degradation, the tissue can be treated according to themethod of the present invention. For example, the tissue can be immersedin a saline solution (preferably, normal saline at a concentration ofabout 0.8% to about 1.0% by weight) containing about 20 millimolar (mM)to about 30 mM EDTA or EGTA, about 10 mM to about 30 mM HEPES, and aconcentration of sodium hypochlorite (5% stock bleach solution) of noless than about a 1:400 dilution or no more than about 1:50 of stockbleach (which generally corresponds to about 2 mM to about 20 mM). Thetissue preferably remains immersed in the bleach solution for a periodof no less than about 24 hours, and more preferably, no more than about36 hours, at room temperature.

[0018] Prior to or subsequent to contacting the tissue with thecomposition containing the oxidizing agent, the tissue is preferablycontacted with a detergent composition. More preferably, this includescontacting the tissue with a first detergent composition and then asecond detergent composition. Preferably, the first detergentcomposition includes an ionic detergent and the second detergentcomposition includes a nonionic detergent. Preferably, these steps arecarried out at a temperature of at least about 30° C. In certainembodiments, these steps involve sonicating the tissue while in thedetergent compositions at a temperature of about 30° C. to about 45° C.

[0019] Proteins suspected of being involved in the initial events ofcalcification are not necessarily accessible due to their tertiarystructure, which may hide groups. Therefore, the use of reducing agentsmay be employed because they can be effective in inactivating proteinswith biological activity. Thus, for certain preferred embodiments, thedetergent composition can include a reducing agent such as DTT andothers that are capable of reducing disulfide bonds.

[0020] Another preferred embodiment is directed to a method for reducingmineralization of a tissue-derived implantable medical device. Themethod involves: contacting the tissue with a composition including atleast one oxidizing agent; rinsing the tissue to remove at least aportion of the oxidizing. agent; and contacting the tissue with adetergent composition including at least one reducing agent prior toimplantation of the medical device.

[0021] Yet another preferred embodiment is directed to a method forreducing mineralization of a tissue-derived implantable medical device.The method involves: contacting the tissue with a non-phosphate bufferedorganic saline solution; contacting the tissue with a compositionincluding at least one oxidizing agent; rinsing the tissue to remove atleast a portion of the oxidizing agent; contacting the tissue with afirst detergent composition including at least one ionic detergent andat least one reducing agent; rinsing the tissue to remove at least aportion of the first detergent composition; and contacting the tissuewith a second detergent composition including at least one nonionicdetergent and at least one reducing agent prior to implantation of themedical device.

[0022] The compositions used in the present invention are all typicallyaqueous based. Furthermore, the treatment steps of the present inventioncan be carried out in any order desired. For example, tissue can betreated with a composition containing an oxidizing agent followed by oneor more detergent compositions, with or without a reducing agent,followed by fixing the tissue. Alternatively, tissue can be treated witha detergent composition, with or without a reducing agent, followed byan oxidizing agent, followed by a second detergent composition, with orwithout a reducing agent, followed by fixing. Or, the fixation process(using a glutaraldehyde- or a carbodiimide-based process, for example)can be carried out first, e.g., prior to contacting the tissue with anoxidizing agent or a detergent composition.

[0023] As used herein, a “tissue-derived implantable medical device” ismeant to include an organ or tissue that is derived in whole or partfrom an animal, typically a mammal, or which is made from other organictissue and is to be implanted alone or as part of a bioprostheses. Thus,the term generally includes bioprosthetic tissue, such as hearts, heartvalves, aortic root tissue and other heart components, pericardium,vascular replacements, e.g., veins and/or arteries, or grafts, heartreplacements, urinary tract and bladder replacements, bowel and tissueresections in general, and the like.

[0024] As used herein, the term “pathologic calcification” refers to theundesirable deposition of calcium phosphate mineral salts. Without beingbound by any theory or mechanism, calcification may be due to hostfactors, implant factors, and other patient-related extraneous factors.There is evidence to suggest that deposits of calcium are related todevitalized cells and, in particular, cell membranes having calciumchannels that are no longer functioning or are malfunctioning.Calcification has been observed to begin with an accumulation of calciumand phosphorous, present under the appropriate conditions ofconcentration and molecular alignment to form hydroxyapatite, whichdevelops into nodules that can eventually lead to valvular failure in atissue-derived implantable device.

[0025] As used herein, the term “reduced mineralization” refers to aquantitative decrease in the observed accumulation of minerals, such ascalcium phosphate mineral salts, to a tissue-derived implantable medicaldevice of the invention and refers preferably to the aortic walls andleaflets of such devices.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0026] 1. Procurement and Initial Treatment of Tissue.

[0027] A tissue-derived implantable medical device employed in a methodof the invention can be obtained from a mammalian species. Mammalianspecies suitable for providing tissue for a tissue-derived implantablemedical device in the invention, include, for example, pigs, cows,sheep, etc. Preferably, the mammalian species is a pig or a cow.Preferred tissues for use in the present method include, for example,porcine aortic root tissue, bovine aortic root tissue, porcine and/orbovine pericardium or veins and arteries. Preferably, the tissue-derivedimplantable medical device contains a heart valve.

[0028] Typically, the tissue for a tissue-derived implantable medicaldevice is obtained directly from a slaughter house, dissected at theslaughter house to remove undesired surrounding tissue. Either at thesite of slaughter or shortly thereafter prior to significant tissuedamage and/or degradation, the tissue is treated according to thepresent invention. It can be treated by the various steps of theinvention in any of a wide variety of orders.

[0029] In a typical situation, once the tissue is obtained it is shippedon ice in order to reduce autolytic damage to the tissue and to minimizebacterial growth during shipment. Preferably, the tissue is shipped andreceived within about 24 hours to a location where treatment of thetissue, as described herein, can be performed.

[0030] In one method, the tissue is thoroughly rinsed with anon-phosphate buffered organic saline solution. The non-phosphatebuffered organic saline solution stabilizes the tissue matrix whileassisting in the removal of excess blood and body fluids that may comein contact with the tissue. A non-phosphate buffered organic salinesolution is preferred in the present method as it serves to removephosphate containing material in the tissue-derived implantable medicaldevice.

[0031] Suitable buffering agents for the non-phosphate buffered organicsaline solution used in the practice of the invention are thosebuffering agents which have a buffering capacity sufficient to maintaina physiologically acceptable p,H and do not cause deleterious effects tothe tissue-derived implantable medical device. Preferably, thenon-phosphate buffered organic saline solution includes a bufferingagent in a concentration of about 10 mM to about 30 mM. Buffering agentsinclude, for example, acetate, borate, citrate, HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), BES (N,N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid), TES(N-tris[Hydrpxymethyl]methyl-2-aminoethanesulfonic acid), MOPS(morpholine propanesulphonic acid), PIPES(piperazine-N,N′-bis[2-ethane-sulfonic acid]), or MES (2-morpholinoethanesulphonic acid), and typically provides buffering in a pH range ofabout 6.5 to about 8.5. An organic buffer is preferred as it willtypically not add additional phosphate to the tissue matrix, which mayparticipate in the formation of hydroxyapatite as do other physiologicbuffers known in the art, such as sodium phosphate. Organic buffers alsoprovide a means of buffering solution without interfering withsubsequent crosslinking chemistry. Preferably, the buffering agent isHEPES, TES, BES, MOPS, PIPES, or MES. More preferably, the bufferingagent employed in a saline solution of the invention is HEPES, as itprovides a pKa of about 7.4, which is very suitable for tissueprocessing.

[0032] Employing a non-phosphate buffered organic saline solutiontypically decreases the likelihood of a nucleation event. Using phospatesalts to buffer solutions may increase the levels of phospate, PO₄ ³⁻,to the point that it will bind available divalent cations such ascalcium, thus creating an environment prone to precipitate calciumphospate salts. Excessively high phosphate and calcium levels have beenused in in vitro calcification models. Depriving such matrices of theseelements will forestall early nucleation events from occurring duringtissue processing procedures, as nucleation may have long term effectsafter the tissue is fixed and stored prior to implantation. Thus,preferably, in a first treatment step a dissected tissue is treated asquickly as possible, typically within 24 hours, with a non-phosphatebuffered organic saline solution of the invention. Early treatment ofthe tissue is preferred as this allows better diffusion of soluble ionssuch as calcium, phospate, magnesium, divalent cations, and anioniccompounds, in general, out of the tissue. These elements are primarycomponents of dystrophic mineralization, in general, and calcification,specifically.

[0033] The non-phosphate buffered organic saline solution suitable foruse in the present invention typically contains additional components,which include, for example, a saline solution, preferably, of about 0.8%to about 1.0% by weight. Additionally, the non-phosphate bufferedorganic saline solution preferably contains a chelating agent. Thechelating agent is preferably present in the solution at a concentrationof about 20 mM to about 30 mM. Suitable chelating agents include, forexample, EDTA (ethylenediaminetetraacetic acid), EGTA(ethylenebis(oxyethylenenitrilo)tetraacetic acid),

[0034] ethylenebis(oxyethylenenitrilo)tetraacetic acid, citric acid, orsalts thereof, and sodium citrate. A chelating agent employed in amethod of the invention preferably binds divalent cations, such ascalcium, magnesium, zinc, and manganese. Removal of such ions from thetissue-derived implantable medical device during initial processing mayrender the tissue less susceptible to spontaneous precipitation of thesedivalent ions with phosphate ions that may be present in the tissue.Thus, taking away these divalent cations will prevent apatite formation.

[0035] In a preferred embodiment, the non-phosphate buffered organicsaline solution of the invention is about 0.9 wt-% saline, buffered to apH of about 7.4 with about 10 (millimolar) mM to about 30 mM HEPESbuffer and contains about 20 mM to about 30 mM of EDTA. Subsequent torinsing in the non-phosphate buffered organic saline solution, asdescribed above, the tissue-derived medical device may be maintained atabout 4° C. for about 4 hours to about 16 hours until furtherprocessing.

[0036] 2. Subsequent Treatment, Transport, and Storage of the Tissue.

[0037] After the tissue-derived implantable medical device has beenrinsed and stored prior to shipment, the procured tissue is preferablytransferred and immersed in a composition containing an oxidizing agent.Alternatively, the tissue can be immediately rinsed and stored in acomposition containing an oxidizing agent. This composition can be usedto transport the tissue from the slaughterhouse to the manufacturingfacility.

[0038] The composition includes one or more oxidizing agents. Typically,oxidizing agents are capable of accepting electrons, although in thisparticular system they may not be functioning in that capacity.Preferably, the oxidizing agent is selected from the group of sodiumhypochlorite, sodium bromate, sodium hydroxide, sodium iodate, sodiumperiodate, performic acid, periodic acid, potassium dichromate,potassium permanganate, chloramine T, peracetic acid, and combinationsthereof. More preferably, the oxidizing agent is selected from the groupof sodium hypochlorite, performic acid, periodic acid, peracetic acid,and combinations thereof. The oxidizing agent is preferably in thecomposition in an amount of about 2 mM to about 20 mM, and morepreferably, about 5 mM to about 10 mM.

[0039] The composition also preferably includes a chelating agent, asdescribed above, and also preferably includes a buffering agent and asaline solution in the concentrations described above. Preferably, thebuffering agent has a pKa of about 6.5 to about 7.5. More preferably,the buffering agent is an organic buffer, such as HEPES, TES, BES, orothers as described above. Preferably, the tissue is immersed in a 0.9%saline solution (normal saline) containing about 20 mM to about 30 mMEDTA, about 10 mM to about 30 mM HEPES, and a concentration of sodiumhypochlorite (5% stock bleach solution) of no less than about a 1:400dilution or no more than about 1:50 of stock bleach (which generallycorresponds to about 2 mM to about 20 mM). The tissue preferably remainsimmersed in the bleach solution for a period of no less than about 24hours, and more preferably, no more than about 36 hours, at roomtemperature.

[0040] The composition containing an oxidizing agent, such as sodiumhypochlorite may act to “inactivate” the activity of specific proteinssuch as kinases, phospatases and others within the tissue matrices thatmay be essential for the initiation of the calcification process. It isthought that the use of oxidizing agents in the composition may inhibitbiological functions of protein structure. For example, the oxidation ofspecific R groups of amino acid side chains may interrupt the folding ofspecific proteins, thus disrupting the native confirmation of theenzyme, rendering if unable to process substrate.

[0041] Although not wishing to be bound by theory, it is believed thatthe oxidizing agent uncouples enzymatic reactions that occur withincells that reside in the extra-cellular matrix (ECM). These enzymesutilize calcium and phosphate in a variety of energy utilization andcell signaling mechanisms. Under the proper conditions, that may bepresent during tissue processing, these enzymes may be responsible forsequestering the ions necessary to form apatite (i.e., hydroxyapatite)in the ECM. Other reactions can take place besides the modification ofprotein active sites. Disulfide bonds that may exist in specificproteins may be broken, resulting in protein dysfunction by altering itstertiary structure.

[0042] Compositions containing one or more oxidizing agents may work onreducing sugars present in glycosaminoglycans such as heparin,chondroitin sulfate, dermatin sulfate. The oxidative process could yieldthese compounds susceptible to crosslinking. This solution can also beconsidered a bacteriocidal solution and will inhibit the growth ofbacteria in the solution during transport. A tissue-derived implantablemedical device placed in the composition containing an oxidizing agentdescribed above, can be stored and/or shipped as desired.

[0043] 3. Additional Processing of the Tissue.

[0044] Preferably, subsequent to treatment with the compositioncontaining an oxidizing agent as described above, the tissue-derivedimplantable medical device is rinsed exhaustively with a non-phosphatebuffered organic saline solution described above to completely removethe oxidizing agents. Preferably, the tissue-derived implantable medicaldevice is rinsed at room temperature for about 8 to about 18 hours.Preferably, the non-phosphate buffered organic saline solution ischanged frequently, e.g., about every 20 to about 30 minutes. Thefrequent solution change can equate to about a 32-volume change, i.e.,approximately 250 mL per volume change, during the course of the rinsetreatment.

[0045] Regardless of the specific rinse treatment protocol employed, thetissue to non-phosphate buffered organic saline solution ratio, i.e.,tissue to volume ratio, should be fairly large. A large tissue to volumeratio employed in the method is designed-to create the largest possiblegradient for solute diffusion (i.e., removal) out of the tissue ECM andfacilitate movement of materials into the surrounding non-phosphatebuffered organic saline solution. The frequent volume changes areeffective in maintaining the diffusion gradients to assist in theremoval of the oxidizing agent from the ECM. During the rinse treatmentdescribed herein, the tissue may optionally be subjected to ultrasonicprocessing using a sonicator. Employing a sonicator may be advantageousin that it may further assist in the diffusion of materials from thetissue.

[0046] Additionally, the temperature during the rinse treatmentdescribed above is preferably maintained at less than about 45° C.Maintaining the temperature of the rinse treatment can be accomplishedby employing a heat exchange system in combination with the ultrasonicprocessing. The heat exchange system involves pumping the detergentcomposition from a reaction vessel (containing the tissue) into astainless steel coil immersed in a water bath (heat exchanger),preferably at a temperature of about 35° C. to about 45° C., and backinto the reaction vessel.

[0047] 4. Treatment of the Tissue with a Detergent.

[0048] Preferably, after the tissue is thoroughly rinsed, thus ensuringremoval of the oxidizing agent, the tissue is immersed in a firstcomposition containing at least one detergent. Preferably, the detergentis an ionic detergent such as sodium dodecyl sulfate (SDS), althoughnumerous other ionic detergents can be used. Examples include sodiumcaprylate, sodium deoxycholate, and sodium 1-decanesulfonate. Theconcentration of ionic detergent is preferably within a range of about0.5% to about 2.5% (weight by volume for solids or volume by volume forliquids), and more preferably, about 0.5% to about 1.5% (weight byvolume for solids or volume by volume for liquids). The detergentcomposition preferably also contains about 10 to about 30 mM HEPES (orother buffer as described above), about 20 mM to about 30 mM EDTA (orother chelating agent as described above), and saline in an amount ofabout 0.8% to about 1.0% (by weight).

[0049] The solution may also optionally contain a reducing agent such asDTT (dithiothreotol) (or similar such agents) in a range of 10 MM toabout 200 mM. Examples of other suitable reducing agents include, forexample, 2-mercaptoethylamine and DTE (dithioerythritol). Reducingagents also have the potential lo inactivate proteins and such treatmentalters the tertiary structure of bioactive compounds and makes theminactive. Alternatively, the tissue can be treated with an aqueoussolution containing the reducing agent and other optional componentswithout a detergent.

[0050] The tissue is subsequently placed in the first detergentcontaining composition for a period of at least about 24 hours at atemperature of at least about 30° C. The temperature at which thisprocess takes place can have a significant effect on the ability ofdetergents to gain access to phospholipids of the cell membranes andassociated proteins to be removed or modified. It is known that whenphospholipid bilayers are heated they undergo changes in physicalproperties over specific temperature ranges. This “phase transition” isdue to the increased motion about the carbon-carbon bonds of fatty acylchains. The acyl chains pass from a highly ordered, gel-like state atcooler temperatures (i.e., room temperature) to a more mobile fluidstate at higher temperatures. During the gel-to-fluid transition,thermal energy is absorbed and the bilayer passes through the “meltingtemperature” of the bilayer. The fluidity of the membrane bilayers atthe temperatures used in this process allow for greater ease ofdissolution of the cell membranes.

[0051] During the treatment of the tissue with a detergent, the tissuemay be subjected to ultrasonic processing as described above. Thetemperature during this treatment is typically maintained at less thanabout 50° C., and preferably, less than about 45° C., using a heatexchange system in combination with the ultrasonic system, for example.

[0052] The detergent is used to facilitate the removal of cells, celldebris, and cell organelles from the ECM. Cell membranes and cellorganelle membranes are known to house a large part of the enzymes andproteins implicated in the nucleation of apatite formation during themineralization process. Solubilization of these membranes may alsoassist in the inhibition of calcification by denaturing the proteinsduring the extraction process. It has also been proposed thatphospholipids, which make up the largest portion of cell membranes, areinvolved in the initiation of calcification. Detergent treatments willbreak up the phospholipid bilayer of cell membranes in the process ofextracting the proteins.

[0053] After treatment with the detergent composition, thetissue-derived implantable medical device is typically processed throughanother exhaustive rinse process utilizing the non-phosphate bufferedorganic saline solution described above. After rinsing, thetissue-derived implantable medical device is placed into a seconddetergent composition. The second detergent composition preferablycontains a greater affinity for phospholipids than the first detergentcomposition discussed above. Preferably, the second detergentcomposition contains the detergent NP-40, although other nonionicdetergents can be used such as Triton X-100, Tween series, andoctylglucoside. The use of this detergent is designed to further assistin the removal of cellular material and debris.

[0054] This second detergent composition preferably also contains about10 to about 30 mM HEPES (or other buffer as described above), about 20mM to about 30 mM EDTA (or other chelating agent as described above),and saline in an amount of about 0.8% to about 1.0% (by weight). Thisdetergent may also have a reducing agent as described above in the firstdetergent composition. The detergent concentrations employed in thesecond detergent containing composition are similar to the detergentconcentrations employed in the first detergent containing composition,with the standard concentration used preferably being about 0.5% toabout 2.5% (volume by volume for liquids), and more preferably about0.5% to about 1.5% (volume by volume for liquids). The nonionicdetergents described herein may act in different ways at varyingconcentrations. For example, at high concentrations (above the criticalmicelle concentration) nonionic detergents solubilize biologicalmembrane by forming mixed micelles of detergents, phospholipid, andintegral membrane proteins. At low concentrations, nonionic detergentsmay bind to the hydrophobic regions of most membrane proteins, makingthem soluble in aqueous solutions.

[0055] Typically, the tissue-derived implantable medical device isplaced in the second detergent containing composition for a period of atleast about 24 hours at a temperature of at least about 30° C. Duringthis stage of the process, the tissue-derived implantable medicaldevice, as described above, may be subjected to ultrasonic processing.As further described above, the temperature is typically maintained atless than about 50° C., and preferably, less than about 45° C., using aheat exchange system in combination with the ultrasonic system.

[0056] After treatment with the second detergent containing composition,the tissue-derived implantable medical device is rinsed exhaustively inthe non-phosphate buffered organic saline solution described above forabout 24 hours at room temperature (e.g., about 25° C. to about 30° C.).At the completion of the rinse treatment, the tissue-derived implantablemedical device may be placed in the non-phosphate buffered organicsaline solution and stored at 4° C. until the fixation process.

[0057] One of skill in the art will appreciate that the order of the useof the detergent containing compositions may be changed. That is,treatment of a tissue-derived implantable medical device with a nonionicdetergent containing composition can be used prior to treatment of thetissue-derived implantable medical device with an ionic detergentcontaining composition. Moreover, the method described herein isspecifically designed to be modular, in that particular treatment stepscan be placed anywhere within the tissue-derived implantable medicaldevice processing procedure. For example, a detergent may be used in atransport solution prior to the exposure of the oxidizing agents,followed by another detergent treatment.

[0058] 5. Fixation of the Tissue.

[0059] Preferably, after detergent treatment and rinsing treatment asdescribed above, fixation of the tissue, although fixation could occurbefore any treatment steps described herein. Typically, prior tofixation, the tissue-derived implantable medical device is rinsed with anon-phosphate buffered organic saline solution similar to thenon-phosphate buffered organic saline solution described above, however,the buffering agent, such as HEPES, is employed at concentration ofabout 10 mM to about 20 mM. Two alternative fixation treatments topreserve the tissue-derived implantable medical device can be employedin the method of the present invention. A first fixation treatmentemploys a cross-linking process which introduces the tissue-derivedimplantable medical device to about a 0.2% glutaraldehyde solution inthe prepared non-phosphate buffered organic saline solution describedabove. This fixation process takes approximately 7 days to complete, andis well known to one of skill in the art.

[0060] A second fixation treatment employs a cross-linking process whichintroduces the tissue-derived implantable medical device to about awater soluble carbodiimide as disclosed in U.S. Pat. Nos. 5,447,536(Giardot et al.) and 5,733,339 (Giardot et al.) and EP 897942 A (Cahalanet al.). This fixation process is a two-stage process that utilizes moreof the available side groups on the amino acid backbone of the collagenmolecules.

[0061] The following examples more fully describe the present invention.Those skilled in the art will recognize that the particular reagents,equipment and procedures described are merely illustrative and are notintended to limit the present invention in any manner.

EXAMPLE

[0062] Enzyme Activity Related to Various Alternative Tissue ProcessTreatments

[0063] I. Background

[0064] Current treatments for the inhibition of calcification do notoffer total protection for both leaflet and wall. Based on publishedreports in the literature the initiation events of calcification aremost certainly cellular based. The exact mechanism for this nucleationevent is unknown but may involve proteins that are involved in the useof calcium and/or phosphate in signal transduction, energy utilization,post transnational modifications of proteins or ion balance within acell.

[0065] In an attempt to understand if these first steps could beinvolved in nucleation events, proteins were used to test the theory ofinactivating their function by denaturation. The model system utilizesproteases to test the function of protein after treatment. Theseproteins are assisted in maintaining their tertiary structure bydifferent bonding mechanisms, trypsin by hydrogen bonds and chymotrypsinby a combination of disulfide bonds and hydrogen bonds.

[0066] In these experiments trypsin and chymotrypsin were chosen basedon their ability to digest a collagen based substrate (AZOCOLL, SigmaChemical) that liberates a colored byproduct after enzymatic attack.

[0067] II. Summary

[0068] Trypsin and chymotrypsin were subjected to severaldenaturation/inactivation methodologies, either by exposure to oxidizingagents, reducing agents, or detergent(s). The preliminary data indicatesthat denaturing can inactivate these proteins by modifying theirtertiary structure.

[0069] III. Materials

[0070] Phosphate Buffered Saline (PBS), Sigma product no. 1000-3, SigmaChemical Co., St. Louis, Mo.

[0071] Normal Saline (NS), Sigma 430AG-4, Sigma

[0072] AZOCOLL (Azo dye impregnated collagen, product no. 194933)Calbiochem, San Diego, Calif.

[0073] Sodium hypochlorite (Bleach)

[0074] Peracetic acid, Aldrich Chemical Co., Milwaukee, Wis.

[0075] SDS, Sigma product no. L 4509, Sigma Chemical

[0076] Trypsin (Type I, bovine pancreas, product no. T 4665), Sigma

[0077] Chymotrypsin (bovine pancreas, product no. C 4129), Sigma

[0078] DTT (dithiothreatol, product no. D 0632), Sigma

[0079] Water bath

[0080] Spectrophotometer (Beckman Model)

[0081] IV. Methods

[0082] Protein Preparation

[0083] Because of the sensitivity of these enzymes to auto-digestion allprotein solution were made fresh on the day of the experiment and storedon ice until use.

[0084] Stock trypsin and chymotrypsin solutions were solubilized fromdry powder supplied by the manufacturer in PBS, pH 7.4, at roomtemperature. Protein concentrations were set at 1 mg/ml. Proteinsolutions were sterile filtered through a 0.45 μm syringe filter toeliminate potential bacterial contamination.

[0085] Working solution of the protein was prepared by dilution fromconcentrated stock to a final concentration of 100 μg/ml.

[0086] Protein Treatment

[0087] Each of the proteins was exposed to various agents under thefollowing conditions.

[0088] Oxidizing agents: Proteins were exposed to either peracetic acidor bleach for a period of 2 hours at room temperature. After treatmentthe proteins were passed over a desalting column (Sephadex G-25) toseparate the oxidizing agent from the protein. Protein was eluted fromthe column using PBS. Elution of the protein was tracked by monitoringUV absorbance at 280 nm. The eluted protein was harvested in 0.5 mlaliquots. The aliquots that had the highest UV absorbance were used forthe analysis.

[0089] Reducing agents: Chymotrypsin was exposed to DTT (finalconcentration 10 mM PBS, pH 7.4) in the presence of SDS (1% w:v). Theprotein was incubated with the agents for 2 hours. In order to assureall disulfide bonds were acted on and fully reduced, the reaction wasperformed at elevated temperature (37° C.-40° C.). The protein waspassed over a Sephadex G-25 column, as described above, to prepare itfor the assay. After the protein was eluted from the column it was usedimmediately in the assay.

[0090] Denaturing: Trypsin was exposed to SDS (final concentration 1%w:v in PBS, pH 7.4). The protein was prepared for the assay by dialyzingagainst PBS to remove excess SDS. Dialysis was performed at 4° C. tominimize auto-digestion.

[0091] Enzyme Activity Assessment

[0092] Each of the proteins that were exposed to an agent was assessedfor its activity by incubating it with a collagen substrate with an azocontaining dye. AZOCOLL was suspended in PBS at a concentration of 1mg/ml.

[0093] For the assay, 1 ml of protein was mixed with 1 ml of AZOCOLLsubstrate. The reaction was performed at 37° C. for 20 minutes inEpendorff microfuge tubes. At the end of the reaction time, the reactionwas terminated by; centrifuging the mixture, separating the undigestedAZOCOLL from the aqueous solution. The aqueous solution was removed fromthe centrifuge tubes and the absorbance of the solution (containingsoluble azo dye) was monitored at 520 nm.

[0094] Controls consisted of a negative control, which was PBScontaining the appropriate agent employed to act on the protein, butwithout the protein itself. Positive controls were enzymes that were notexposed to the treatments. They were aliquoted directly from the stocksolution into the AZOCOLL reaction vessel.

[0095] Data Analysis

[0096] All experiments were run in triplicate. Data reported is the meanof the 3 readings. The experiments were done to test feasibility of themodel system; no attempt was made to do statistical analysis on orbetween the various groups.

[0097] V. Results Trypsin exposure to oxidizing agent(s) Sample Abs. @520 nm Negative control 0.089 Positive control 0.664 Treated sample0.342

[0098] Trypsin exposure to denaturing agent (SDS) Sample Abs. @ 520 nmNegative control 0.004 Positive control 0.544 Treated sample 0.066

[0099] Chymotrypsin exposed to reducing agent Sample Abs @ 520 nmNegative control 0.003 Positive control 0.523 Treated sample 0.031

[0100] VI. Discussion

[0101] The data suggests that the treatments above may be helpful ininactivating proteins within the extracellular matrix of tissue. Thereare several points that need to be evaluated before a model is adoptedand considered reliable for evaluating subsequent tissue matrixtreatments. The first is the appearance of reacted substrate in thenegative controls. This may mean that, in the case of oxidizing agentsthat there is some possible reaction occurring by association of thereagents.

[0102] For the other experiments utilizing SDS, the low backgrounds maybe due to excess SDS that is carried into the assay from the enzymeexposed to the reaction. The SDS may bind to the AZOCOLL making it lesssusceptible to degradation.

[0103] All publications, patents and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method of making a tissue-derived implantablemedical device, the method comprising contacting the tissue with acomposition comprising at least one oxidizing agent prior toimplantation of the medical device.
 2. The method of claim 1 furthercomprising rinsing the tissue to remove at least a portion of theoxidizing agent.
 3. The method of claim 1 wherein the tissue is obtainedfrom a mammal.
 4. The method of claim 1 wherein the tissue-derivedimplantable medical device is rendered resistant to in vivo pathologiccalcification.
 5. The method of claim 1 wherein the tissue-derivedimplantable medical device comprises a heart valve.
 6. The method ofclaim 5 wherein the heart valve is derived from porcine aortic roottissue, bovine aortic root tissue, porcine pericardium, bovinepericardium, bovine veins, porcine veins, bovine arteries, or porcinearteries.
 7. The method of claim 1 wherein the oxidizing agent isselected from the group of sodium hypochlorite, sodium bromate, sodiumhydroxide, sodium iodate, performic acid, sodium periodate, periodicacid, potassium dichromate, potassium permanganate, chloramine T,peracetic acid, and combinations thereof.
 8. The method of claim 7wherein the oxidizing agent is selected from the group of sodiumhypochlorite, performic acid, periodic acid, peracetic acid, andcombinations thereof.
 9. The method of claim 8 wherein the sodiumhypochlorite is in a concentration of about 2 mM to about 20 mM.
 10. Themethod of claim 1 wherein the composition comprising an oxidizing agentfurther comprises at least one chelating agent.
 11. The method of claim10 wherein the chelating agent is selected from the group ofethylenediaminetetraacetic acid,ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), citric acid,salts thereof, and combinations thereof.
 12. The method of claim 1wherein the composition comprising at least one oxidizing agent furthercomprises at least one buffering agent.
 13. The method of claim 12wherein the buffering agent has a pKa of about 7.0 to about 7.5.
 14. Themethod of claim 12 wherein the buffering agent is an organic buffer. 15.The method of claim 14 wherein the buffering agent is selected from thegroup of HEPES, TES, BES, MOPS, PIPES, MES, and combinations thereof.16. The method of claim 1 wherein contacting the tissue with acomposition comprising at least one oxidizing agent is carried out forat least about 24 hours.
 17. The method of claim 1 further contactingthe tissue with a detergent composition.
 18. The method of claim 17wherein the detergent composition comprises at least one reducing agent.19. The method of claim 17 wherein the detergent composition comprisesan ionic detergent or a nonionic detergent.
 20. The method of claim 17wherein contacting the tissue with the detergent composition is carriedout at a temperature of at least about 30° C.
 21. The method of claim 17wherein contacting the tissue with the detergent composition comprisessonicating the tissue while immersed in the composition.
 22. The methodof claim 17 wherein contacting the tissue with a detergent compositioncomprises contacting it with a first and a second detergent composition.23. The method of claim 22 wherein the first detergent compositioncomprises an ionic detergent and the second detergent compositioncomprises a nonionic detergent.
 24. The method of claim 23 wherein thefirst and/or the second detergent compositions comprise at least onereducing agent.
 25. The method of claim 17 wherein contacting the tissuewith a detergent composition is carried out after contacting the tissuewith the composition comprising the oxidizing agent.
 26. The method ofclaim 1 further comprising treating the tissue with a fixativecomposition.
 27. The method of claim 26 wherein treating the tissue witha fixative composition comprises using a glutaraldehyde- or acarbodiimide-based process.
 28. The method of claim 27 wherein thefixative composition further comprises at least one buffering agent. 29.A method for reducing mineralization of a tissue-derived implantablemedical device, the method comprising: contacting the tissue with acomposition comprising at least one oxidizing agent; rinsing the tissueto remove at least a portion of the oxidizing agent; and contacting thetissue with a detergent composition comprising at least one reducingagent prior to implantation of the medical device.
 30. A method forreducing mineralization of a tissue-derived implantable medical device,the method comprising: contacting the tissue with a non-phosphatebuffered organic saline solution; contacting the tissue with acomposition comprising at least one oxidizing agent; rinsing the tissueto remove at least a portion of the oxidizing agent; contacting thetissue with a first detergent composition comprising at least one ionicdetergent and at least one reducing agent; rinsing the tissue to removeat least a portion of the first detergent composition; and contactingthe tissue with a second detergent composition comprising at least onenonionic detergent, and at least one reducing agent prior toimplantation of the medical device.